Reaction products of 4-amino-5-hydroxynaphthalene-2,7-disulfonic acid, coupled twice with diazotized 2-[(4-aminophenyl)sulfonyl]ethyl hydrogen sulfate, sodium salts

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14. Dec. 1987 to 30. Sep 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

absorption

distribution

excretion

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

Deviations:

yes

Remarks:

metabolism determined in separate study

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

bisphenyl-U-14C

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hoechst AG
- Age at study initiation: 7 to 8 weeks
- Weight at study initiation: 160 to 230 g
- Fasting period before study: -
- Housing: single (excretion) 2/cage (plasma levels, exhalation)
- Individual metabolism cages: yes (excretion) / no (plasma levels, exhalation)
- Diet: Altromin 1321 ad libitum
- Water: tap ad libitum
- Acclimation period: -


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 26
- Humidity (%): 30 to 50
- Air changes (per hr): -
- Photoperiod (hrs dark / hrs light): -


IN-LIFE DATES: From: 14. Dec To: 24. Dec. 1987
23. Sep. To: 26. Sep. 1988

Route of administration:

other: oral gavage and intravenous

Vehicle:

other: water and NaCl

Details on exposure:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): - oral: 10 mg/kg bw
- IV: 1 mg/kg bw
- concentration (if solution): - oral: 2 mg/g in aqua bidest
- IV: 0.3 mg/g in NaCl solution

Duration and frequency of treatment / exposure:

single dose

Remarks:

Doses / Concentrations:
p.o.: 10 mg/kg body weight (nominal) - Concentration: 2 mg/g solution
i.v.: 1 mg/kg body weight (nominal) - Concentration: 0.3 mg/g solution

No. of animals per sex per dose / concentration:

Exhalation: 2
Plasma levels: 5
Excretion/remaining concentration p.o.: 5
Excretion i.v.: 3

Control animals:

other: one rat

Positive control reference chemical:

not examined

Details on dosing and sampling:

- Tissues and body fluids sampled: urine, faeces, blood, plasma, exhalate, tissues (spleen, stomach, small intestines, liver, kidneys, gonads, heart, lungs, skeletal muscle, subcutaneous fat, retroperitoneal fat, brain, eyes)
- Time and frequency of sampling:
- Blood sampling (tip of tail): 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 24, 32, 48, 72, 96, 120, 144, 168 h after test item administration
- Urine sampling: in polyethylen bottles 0 -2, 2 -4, 4 -8, 8 -24, 24 -48, 48 -72, 72 -96, 96 -120, 120 -144, 144 -168 h after test item administration
- Feces sampling: in glass vessels: 0 - 24, 24 -48, 48 -72, 72 -96, 96 -120, 120 -144, 144 -168 h after test item administration
- Organ/tissue sampling: directly after sacrifice - 7 days after test item administration
- Exhaled air: continuous aspiration at 0.2 m³/h
- Method type(s) for detection: Liquid scintillation counting
- Limits of detection and quantification: determination of blank value

Statistics:

no data

Preliminary studies:

NA

Details on absorption:

oral: 28.6 %

Details on distribution in tissues:

Blood: 0.25 µg equivalent/mL
Liver: 0.18 µg equivalent/g
Kidneys: 0.10 µg equivalent/g
All other organs: <0.1 µg equivalent/g

In sum 0.28 % of the administered dose; with mean values of 0.13 % in the blood and 0.10 % in the liver

Details on excretion:

Oral administration:
Excretion mainly via feces: 83.71 % of the administered dose
Renal excretion: 14.79 % of the administered dose
Bi-phasic elimination
no excretion by exhalation

Intravenous administration:
Main excretion renal: 51.72 % - bi-phasic
Fecal excretion: 26.5 %

Metabolites identified:

no

Details on metabolites:

see metabolism study HOE 88.1064

Conclusions:

Interpretation of results (migrated information): no bioaccumulation potential based on study results
Incomplete absorption (ca. 28.6 %) after oral administration. About 85 % of the administered dose were excreted via feces and about 15 % via urine. The highest doses of radioactivity were found in blood and liver, 7 days after test item administration (about 0.28 % of the administered dose together with all other organs). After intravenous administration, about 52 % of the radioactivity were found in urine within 3 days after test item administration.

Executive summary:

The kinetics of [14]C-labeled the test substance was investigated in 5 male rats after oral gavage of 10 mg/kg body weight. For assessment of the absorption rate after oral gavage, one group of 3 rats were treated intravenously with 1 mg/kg bw.

The mean absorption rate was 28.6 % - determined by comparison of the renal excretion after oral and IV administration. The slow increase of radioactivity resulted in similar Cmax of 0.7 to 0.8 µg equivalent/g after 5.6 hours in plasma and 21 hours in blood. T1/2 were 5 and 34 hours in plasma and about 8 days in blood, leading to the assumption of of binding of the test substance to, presumably, erythrocytes.

After oral administration, excretion of radioactivity was mainly via feces (80 % to 88 % within 7 days); renal excretion was about 11 % to 18 % in a bi-phasic manner with half-times of 4 to 7 hours and 40 to 69 hours, respectively.

The highest remaining concentration after 7 days was found in blood (0.25 µg eq/mL) and liver (0.18 µg eq/g). Test substance levels in kidneys (0.1 µg eq/g and other tissues were lower. Totally, 0.28 % of the administered dose were found in blood and tissues. The mean over-all recovery rate was 99 % of the administered dose.

Reference 2

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21. Dec. 1987 to 21. March 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

excretion

metabolism

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

Version / remarks:

Tier 1

Deviations:

yes

Remarks:

exhalation determined in separate study

Principles of method if other than guideline:

Determination of radioactivity in the excreta of rats. Identification of metabolites in the excreta.

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

bisphenyl-U-14C

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hoechst AG
- Age at study initiation: -
- Weight at study initiation: 230 g
- Fasting period before study: -
- Housing: 2 per cage
- Individual metabolism cages: no
- Diet: Altromin 1321 ad libitum
- Water: tap ad libitum
- Acclimation period: 1 to 2 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): -
- Photoperiod (hrs dark / hrs light): -

IN-LIFE DATES: From: 21. Dec. 1987 To: 24 Dec. 1987

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg/kg bw
- concentration (if solution): 1.836 mg/g

Duration and frequency of treatment / exposure:

single dose

Remarks:

Doses / Concentrations:
10 mg/kg body weight (nominal), 9.94 mg/kg bw actual dose

No. of animals per sex per dose / concentration:

5 males

Control animals:

no

Positive control reference chemical:

not investigated

Details on study design:

TS was dissolved in bidistilled water (5 minutes ultrasound ) to a final concentration of 1.836 mg/g

Details on dosing and sampling:

- Tissues and body fluids sampled: urine, faeces
- Time and frequency of sampling: 0-24 h, 24-48 h, 48-72 h after dosing
- From how many animals: pooled from all animals
- Method type(s) for identification: Liquid scintillation counting, HPLC-UV, HPLC-14C, TSP-HPLC-MS
- Limits of detection and quantification:
- Other:


TREATMENT FOR CLEAVAGE OF CONJUGATES (if applicable):
- acetylation
- enzyme cleavage with beta-glucoronidase/arylsulfatase

Statistics:

-

Preliminary studies:

not available

Details on absorption:

see kinetic study HOE 89.0388

Details on distribution in tissues:

see kinetic study HOE 89.0388

Details on excretion:

More than 90 % were excreted within the first 24 hours. Excretion mainly via feces.
85 % excretion via feces
15 % excretion via urine
no unchanged test item excreted

Metabolites identified:

yes

Details on metabolites:

Urine: 6 metabolites were separated, two main metabolites identified.
1. 8 % of total radioactivity: sulfate-ester
2. 4 % of total radioactivity: N-acetylate

Feces:
All extractable metabolites identified. About 17% of the radioactivity remained unextracted
1. 76% of total radioactivity: sulfate-ester amount decreased over time
2. N-acetylate increased over time

Conclusions:

Interpretation of results: no bioaccumulation potential based on study results
The test item was almost completely metabolized. The majority of the metabolites were excreted via feces.

Executive summary:

A degradation pathway is proposed based on the results of these studies.

This means that reductive cleavage of the azo groups is the main metabolization step in the rat. The resulting amine is excreted mainly in the feces either directly or after N-acetylation.

Remarkable is the very high excretion rate of the metabolites via feces. Usually such behavior is caused by one of the following acts:

- almost total absorption of the test substance and subsequent biliary excretion of the metabolites

- degradation of the test substance by the intestinal flora

- abiotic hydrolysis of the test substance in the gastro intestinal tract

In this case, the last possibility can be excluded, as the hydrolysis experiments showed products different from those identified as metabolites in the excreta.

The fact that the metabolite pattern in feces is nearly identical with that in urine gives strong evidence that biliary excretion dominates for the test item. However, determination of the absorption rate was done in a further study.

Description of key information

The kinetics and metabolism of the test substance was investigated in studies with radiolabeled substance. Studies revealed that only about 30 % of the substance orally applied is absorbed. About 80 to 88 % of the substance applied was found to be excreted with the faeces within 7 days. Renal excretion was found to be 11 to 18 %. Metabolization of the test substance was also found to take place in the rat while all metabolites detected have been found to be equally or more polar than the mother compound indicating that no bioaccumulation takes place.  

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

28.6

Additional information

The kinetics and metabolism of the test substance was investigated in studies with radiolabeled substance. In the first part, the test substance was labeled at both phenyl rings (bisphenyl-U-¹⁴C), in the second part, in the naphthalene-part. Five male rats received ¹⁴C-labelled HOE CG 0062 as an aqueous solution at a nominal dose of 10 mg/kg body weight by gastric intubation. The reductive cleavage of the azo groups is the main metabolization step in the rat. The resulting amine is excreted mainly in the feces either directly or after N-acetylation.

The very high excretion rate of the metabolites via feces is remarkable. Usually such behavior is caused by one of the following acts:

1. almost total absorption of the test substance and subsequent biliary excretion of the metabolites

2. degradation of the test substance by the intestinal flora

3. abiotic hydrolysis of the test substance in the gastro intestinal tract

In this case, the last possibility can be excluded, as the hydrolysis experiments showed products different from those identified as metabolites in the excreta. The fact that the metabolite pattern in feces is nearly identical with that in urine gives strong evidence that biliary excretion dominates for the test item.

For assessment of the absorption rate after oral gavage, one group of 3 rats were treated intravenously with 1 mg/kg bw. The mean absorption rate was 28.6 % - determined by comparison of the renal excretion after oral and IV administration. The slow increase of radioactivity resulted in similar C_maxof 0.7 to 0.8 µg equivalent/g after 5.6 hours in plasma and 21 hours in blood. T_1/2were 5 and 34 hours in plasma and about 8 days in blood, leading to the assumption of binding of Reactive Black 5 to, presumably, erythrocytes.

After oral administration, excretion of radioactivity was mainly via feces (80 % to 88 % within 7 days); renal excretion was about 11 % to 18 % in a bi-phasic manner with half-times of 4 to 7 hours and 40 to 69 hours, respectively. The highest remaining concentration after 7 days was found in blood (0.25 µg eq/mL) and liver (0.18 µg eq/g). TS levels in kidneys (0.1 µg eq/g) and other tissues were lower. Totally, 0.28 % of the administered dose was found in blood and tissues. The mean over-all recovery rate was 99 % of the administered dose.

In the naphthalene-labeled investigation, urine and feces from 0-24, 24-48 and 48-72 h after application were pooled and investigated using HPLC. Elimination of the administered radioactivity was rapid, quantitative and occurred mainly via feces. 72 h following administration of the dose 109.1 % of the applied radioactivity were recovered in the excreta (108.8 % via feces and 0.3 % via urine). Moreover, nearly 99 % of the dose was excreted in the first 24 h following administration.

No unchanged HOE CG 0062 was detected in urine or feces extract, indicating that rapid and complete metabolization of the test substance took place in the rat. Moreover, all excreted metabolites were even more polar than the test substance itself, which strongly supports the assumption that neither HOE CG 0062 nor any of its metabolites has a bioaccumulation potential. In urine clear separation of at least 3 metabolites (retention times: 3.5-3.8 min; 6.4 min and 13.9-14 min) was achieved and the retention behavior gives strong evidence that each metabolite lost at least one azo-linked side-chain. Moreover, it is very likely that the most polar metabolite (Rt=3.5-3.8 min) has no side-chains at all.

Following extraction with acetonitrile/water, in feces at least 7 metabolites were detected covering retention times from 3 to 23 minutes and containing one main metabolite (Rt=13-15 min). Again, the retention behavior gives strong evidence that the main metabolite has lost at least one azo-linked side-chain. The proposed degradation pathway is given above.

The non-extractable residues in feces accounted for approx. 53 % of the applied dose, indicating that significant amounts of radioactive residues are tightly bound to that matrix. Due to rapid excretion and limited bioavailability of such bound residues, this fraction should be of no toxicological concern. Nevertheless, hydrolysis experiments were performed and revealed a broad peak (Rt=10-20 min) in addition to a main product of very polar character (Rt=3 min), which had most probably lost both side-chains. As it is very likely that cleavage of azo groups will occur during hydrolysis, it may be concluded that the non-extractable residues in feces at least contain the intact naphthalene ring system.

The slow increase of the radioactivity led to concentration maxima of 0.19 and 0.29 μg equivalent/g after on average 6.8 and 6.4 h in blood and plasma, respectively. The concentration decrease occurred with biological half times of 22.1 and 18.5 hours in blood and plasma, respectively. The comparable kinetics in blood and plasma is a hint for the fact that binding of radioactivity to formed blood components does not occur.

After oral gavage, the radioactivity was predominantly eliminated within 7 days via the feces (95.58 ± 1.86 %). The renal (including cage wash) eliminated part was 1.36 ± 0.53 %. Excretion in feces and urine was biphasic. For the first rapid phase, half times of 4.9 h (feces) and approx. 5 h (urine) were calculated or estimated, respectively. In the slow second phase (t½ ca. 75 h for feces and 71 h for urine) only about 1 % (feces) and 0.1 % (urine) or less were excreted.